Enterotoxigenic Escherichia coli strains cause diarrhea in humans and domestic animals by producing two types of enterotoxins, i.e., heat-labile toxin (LT) and heat-stable toxin (ST) (Hofstra et al., 1984, J. Bio. Chem. 259:15182-15187). LT is functionally, structurally, and immunologically related to cholera toxin (CT) (Clements et al., 1978, Infect. Immun. 21:1036-1039). LT and CT are synthesized as holotoxin molecules composed of five identical subunits B and an enzymatically active subunit A (AB5) (Spangler, 1992, Microbio. Rev. 56(4):622-647). The B pentamer binds ganglioside GM1 in the membrane of intestinal epithelial cells or any other cell that contains GM1 (van Heyningen, 1974, Science 183:656-657). Following binding of subunits B to GM1 on the cell surface, subunit A is inserted into cytosal and proteolytically cleaved and reduced at its single disulfide bond to produce an enzymatically active A1 peptide and a smaller A2 peptide (Fishman P H, 1982, J. Membr. Biol. 69:85-97; Mekalanos et al., 1979, J. Biol. Chem. 254:5855-5861; Moss et al., 1981, J. Biol. Chem. 256:12861-12865). The A1 peptide is capable of binding NAD and catalyzing the ADP-ribosylation of Gsα, a GTP-binding regulatory protein associated with adenylate cyclase (Spangler, 1992, Microbio. Rev. 56(4):622-647). The resulting increase in cAMP eventually leads to the release of electrolytes and fluids from affected cells (Cheng et al., 2000, Vaccine 18:38-49).
LT has been shown to function as mucosal adjuvant and induce immune responses against mucosally co-administered antigens (Clements et al., 1988, Vaccine 6:269-277; Elson Colo. 1989, Immunol. Today 146:29-33; Spangler, 1992, Microbio. Rev. 56(4):622-647). However, the high toxicity of wild-type LT has limited its clinical use. Thus, it is desirable to generate mutated LTs having reduced toxicity while retaining immunogenicity.
The term “E. coli heat-labile enterotoxin” or “LT” used herein refers to a heat-labile enterotoxin produced by any enterotoxigenic E. coli strain. The term “E. coli heat-labile enterotoxin subunit A” or “LTA” refers to subunit A of LT. It includes both precursor LTA, which contains a signal peptide, and mature LTA which has the signal peptide removed.